Ion channels activated directly by the binding of cyclic GMP (cGMP) mediate the response to light in vertebrate photoreceptors. Their closure during the sensory transduction event directly couples the chemical signal initiated by isomerization of rhodopsin into changes in membrane potential of the outer segment. Changes in membrane potential in turn direct changes in transmitter release from the synaptic terminal. A binding site for cGMP is present in the carboxyl-terminal domain, and a region that constitutes part of the pore is located between the fifth and sixth transmembrane domains of each subunit of the cGMP-activated ion channel. The molecular mechanism by which cGMP binding leads to opening of the pore, however, is completely unknown. Understanding the structural basis for the function of these channels is critical to understand the role of the channels in visual transduction, and the way its ability to respond to cGMP is tuned under various conditions. Our previous studies have identified two regions of sequence that influence the coupling of cGMP binding to opening of the pore: the amino-terminal region and the cytoplasmic loop that links the sixth (last) transmembrane domain to the cyclic nucleotide-binding domain (post-S6 region).
The specific aims of this study are to examine the molecular mechanism by which these regions communicate binding with cGMP to other parts of the channel, including the pore. We will use electrophysiology, molecular biology, and biochemistry to take a multi-faceted approach to the question of the structural basis for channel function. The tools we have developed for studying the function of and interaction between these two important regions of channel sequence will also be used to study the stoichiometry and architecture of native photoreceptor channels.
